The focus of our research program is to understand the structural basis of muscle contraction at the molecular level, i.e. how the chemical energy of ATP hydrolysis is converted into mechanical energy through a structural transformation. The benefits of the research are not limited to understanding muscle contraction, but the contractile system of muscle can serve as a prototype for many other motile processes in living cells, e.g. cell division and nutrient transport. The basic processes of muscle contraction are well understood: it is a result of cyclic interactions between myosin and actin, driven by the energy of actomyosin ATP hydrolysis. Force is generated by myosin heads (cross-bridges) interacting cyclically with specific sites along the actin filaments. Shortening involves the relative sliding of the myosin filaments and the actin filaments. To explain the muscle movement, the working hypothesis is that some structural changes in the myosin molecule take place while interacting with actin. Since the availability of the crystal structures of the contractile proteins, and with the advent of single molecule assays, the field has made great strides in understanding the underlying processes. However, the details of the energy conversion still remains largely unresolved. One of the obstacles is that most of the studies at the molecular level are based on isolated, in vitro systems. The link between the information obtained from the in vitro systems and the actual processes occurring intact muscle is still largely missing. The aim of our efforts is to provide such a link. X-ray diffraction from permeabilized muscle cells, the technique used in the present study, is one of the few techniques that could reveal in vivo structures in living muscle cells. Although crystal structures of myosin have revealed ligand-dependent differences (e.g. with ATP, ADP or nucleotide free), it is critical to determine if such differences occur in a fully functioning muscle cell. Our results found that major characteristics of the myosin filaments in the muscle cells varied as a function of ligand bound in myosin as well as temperature. In fact, the state of the myosin filaments (ordered or disordered) is directly correlated with the atomic structures of myosin. An ordered thick filament consists of myosins with bound ADP.Pi in a rather rigid and inflexible conformation primed to generate movement when interacting with actin; whereas a disordered thick filament consists of myosin in the other states of ATP hydrolysis where the structure of myosin is more flexible such that their orientation in the thick filament is random. Hence, a link between the microscopic conformation and the macroscopic filament structure has been established: X-ray diffraction patterns can provide a sensitive and relatively simple way of determining the distribution of myosin conformations in living muscle cells. Based on our earlier findings, we have obtained preliminary results in understanding the mechanism of three small-molecule myosin ATPase inhibitors. These inhibitors, BDM, BTS, blebbistatin, greatly reduce the force level generated by the muscle. Our study revealed that these small molecules appear to strongly stabilize ("trap") one of the myosin conformations such that the ATPase cycle cannot be completed. Myosin with these small molecules bound near the active site tends to be more rigid and more compact than the native forms, trapped in the pre-power stroke (myosin.ADP.Pi like) conformation. In fact, the small molecules are so potent in their inhibitory actions that they could partially transform the apo-myosin from its highly flexible and randomly oriented conformation to a more rigid and ordered conformation in the thick filament. The result revealed that even in the absence of nucleotide, myosin can be manipulated to mimic the structural transformation involved in force generation with appropriate ligands. Further significance of this finding is still being probed by theoretical modeling.